Week 10: ANOVA

Goals • Use R to perform analysis of variance (ANOVA) to compare the

means of multiple groups.

• Perform Tukey-Kramer tests to look at unplanned contrasts

between all pairs of groups.

• Use Kruskal-Wallis tests to test for difference between groups

without assumptions of normality.

Learning the Tools

For the examples in this tutorial, we will again return to the Titanic
data set. We’ll group passengers by the passenger class they travelled
under (a categorical variable) and ask whether different passenger
classes differed in their mean age (a numerical variable).
First, load the data.

> titanicData <-

read.csv("DataForLabs/titanic.csv")
Let’s first look at the data to get a sense of how well it fits the
assumptions of ANOVA. Multiple histogram are useful for this
purpose. As we saw last week, we can use ggplot() and facets to
make this plot:

> library(ggplot2)
>
> ggplot(titanicData, aes(x = age)) +
geom_histogram() +
facet_wrap(~ passenger_class, ncol = 1)

1
 These data look sufficiently normal and with similar spreads that
ANOVA would be appropriate.
To confirm these visual impressions, it would be useful to construct a
table of the means and standard deviations of each group. There are
numerous ways to do this in R, but one of the neatest is uses functions
from the package dplyr. If you have not done so yet, install the
dplyr package from the “Packages” tab in RStudio. Then load the
dplyr package with library().

> library(dplyr)

group_by() The package dplyr has several useful features for manipulating data
sets. For our current purposes, we will find two functions particularly
useful: group_by() and summarise(). These two functions are well
named and work together well, first to organize the data by groups,
and second to summarize the results for each group.
First, use group_by() to organize your data frame by the appropriate
grouping variable. For example, here we want to organize the
titanicData by passenger_class:

> titanic_by_passenger_class <- group_by(titanicData,passenger_class)

2
summarise() After applying group_by() to a data frame, we can summarize the
data using summarise(). (The “s” in summarise() is not a typo—the
creator of the package is from New Zealand.) With summarise(), we
can apply any type of function that summarizes data (e.g. mean(),
median(), var(), etc.), and receive that summary group by group.
For example, to calculate the mean age of each passenger_class,
we can use:

> summarise(titanic_by_passenger_class, group_mean

= mean(age, na.rm=TRUE))

# A tibble: 3 × 2
passenger_class group_mean
<fctr> <dbl>
1 1st 39.66777
2 2nd 28.30031
3 3rd 24.51966
As input, we give the name of the grouped table created by
group_by() and the function we want to apply to each group. In this
case we used mean(age, na.rm=TRUE). “group_mean” is a name we
give to that summary variable (it could have been any name we
wanted). The output looks like a table and includes the names of the
groups being summarized. (A “tibble” is not how New Zealanders
spell “table”, but is a type of table like a data frame.) “3 x 2” here
refers to the number of rows x columns in the “tibble” output.
We can give summarise()many summary functions at once, and it will
create columns in the output table for each one. For example, if we
want to output both the mean and the standard deviation, we can
add sd = sd(age, na.rm=TRUE) to the function above.

> summarise(titanic_by_passenger_class, group_mean

= mean(age, na.rm=TRUE), group_sd = sd(age,
na.rm=TRUE))

# A tibble: 3 × 3
passenger_class group_mean group_sd
<fctr> <dbl> <dbl>
1 1st 39.66777 14.93044
2 2nd 28.30031 13.00764
3 3rd 24.51966 11.33059
Note that the standard deviations are very similar, which means that
these data fit the equal variance assumption of ANOVA.

ANOVA Analysis of variance (or ANOVA) is not quite as simple in R as one

3
 might hope. Doing ANOVA takes at least two steps. First, we fit the
ANOVA model to the data using the function lm(). This step carries
out a bunch of intermediate calculations. Second, we use the results
of first step to do the ANOVA calculations and place them in an
ANOVA table using the function anova(). The function name lm()
stands for “linear model”; this is actually a very powerful function that
allows a variety of calculations. One-way ANOVA is a type of linear
model.

lm() The function lm() needs a formula and a data frame as arguments.
The formula is a statement specifying the “model” that we are asking
R to fit to the data. A model formula always takes the form of a
response variable, followed by a tilde(~), and then at least one
explanatory variable. In the case of a one-way ANOVA, this model
statement will take the form
numerical_variable ~ categorical_variable.
For example, to compare differences in mean age among passenger
classes on the Titanic, this formula is
age ~ passenger_class.
This formula tells R to “fit” a model in which the ages of passengers
are grouped by the variable passenger_class.
The name of the data frame containing the variables stated in the
formula is the second argument of lm(). Finally, to complete the
lm() command, it is necessary to save the intermediate results by
assigning them to a new object, which anova() can then use to make
the ANOVA table. For example, here we assign the results of lm() to
a new object named “titanicANOVA”:

> titanicANOVA <- lm(age ~ passenger_class, data = titanicData)

anova() The function anova() takes the results of lm() as input and returns an
ANOVA table as output:

> anova(titanicANOVA)

Analysis of Variance Table

Response: age
Df Sum Sq Mean Sq F value Pr(>F)
passenger_class 2 26690 13344.8 75.903 < 2.2e-16 ***
Residuals 630 110764 175.8
---
Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1

4
 This table shows the results of a test of the null hypothesis that the
mean ages are the same among the three groups. The P-value is very
small, and so we reject the null hypothesis of no differences in mean
age among the passenger classes.

Tukey-Kramer A single-factor ANOVA can tell us that at least one group has a
test different mean from another group, but it does not inform us which
group means are different from which other group means. A Tukey-
Kramer test lets us test the null hypothesis of no difference between
the population means for all pairs of groups. The Tukey-Kramer test
(also known as a Tukey Honest Significance Test, or Tukey HSD), is
implemented in R in the function TukeyHSD().
We will use the results of an ANOVA done with lm() as above, that
we stored in the variable titanicANOVA. To do a Tukey-Kramer test
on these data, we need to first apply the function aov() to
titanicANOVA, and then we need to apply the function TukeyHSD to
the result. We can do this in a single command:

> TukeyHSD(aov(titanicANOVA))

Tukey multiple comparisons of means

95% family-wise confidence level

Fit: aov(formula = titanicANOVA)

$passenger_class
diff lwr upr p adj
2nd-1st -11.367459 -14.345803 -8.389115 0.0000000
3rd-1st -15.148115 -18.192710 -12.103521 0.0000000
3rd-2nd -3.780656 -6.871463 -0.689849 0.0116695
The key part of this output is the table at the bottom. It estimates the
difference between the means of groups (for example, the 2nd
passenger class compared to the 1st passenger class) and calculates a
95% confidence interval for the difference between the corresponding
population means. (“lwr” and “upr” correspond to the lower and
upper bounds of that confidence interval for the difference in means.)
Finally, it give the P-value from a test of the null hypothesis of no
difference between the means (the column headed with “p adj”). In
the case of the Titanic data, P is less than 0.05 in all pairs, and we
therefore reject every null hypothesis. We conclude that the
population mean ages of all passenger classes are significantly
different from each other.

5
Kruskal-Wallis A Kruskal-Wallis test is a non-parametric analog of a one-way ANOVA.
It does not assume that the variable has a normal distribution.
(Instead, it tests whether the variable has the same distribution with
the same mean in each group.)
To run a Kruskal-Wallis test, use the R function kruskal.test(). The
input for this function is the same as we used for lm() above. It
includes a model formula statement and the name of the data frame
to be used.

> kruskal.test(age ~ passenger_class, data =

titanicData)

Kruskal-Wallis rank sum test

data: age by passenger_class

Kruskal-Wallis chi-squared = 116.08, df = 2, p-
value < 2.2e-16
You can see for the output that a Kruskal-Wallis test also strongly
rejects the null hypothesis of equality of age for all passenger class
groups with the Titanic data.

6
7
Questions
1. The European cuckoo does not look after its own eggs, but instead lays them in the
nests of birds of other species. Previous studies showed that cuckoos sometimes have
evolved to lay eggs that are colored similarly to the host bird species' eggs. Is the same
true of egg size -- do cuckoos lay eggs similar in size to the size of the eggs of their
hosts? The data file "cuckooeggs.csv" contains data on the lengths of cuckoo eggs laid
in the nests of a variety of host species. Here we compare the mean size of cuckoo eggs
found in the nests of different host species.

a. Plot a multiple histogram showing cuckoo egg lengths by host species.

b. Calculate a table that shows the mean and standard deviation of length of
cuckoo eggs for each host species.

c. Look at the graph and the table. For these data, would ANOVA be a valid
method to test for differences between host species in the lengths of cuckoo
eggs in their nests?

d. Use ANOVA to test for a difference between host species in the mean size of
the cuckoo eggs in their nests. What is your conclusion?

e. Assuming that ANOVA rejected the null hypotheses of no mean differences,

use a Tukey-Kramer test to decide which pairs of host species are
significantly different from each other in cuckoo egg mean length. What is
your conclusion?

2. The pollen of the maize (corn) plant is a source of food to larval mosquitoes of the
species Anopheles arabiensis, the main vector of malaria in Ethiopia. The production of
maize has increased substantially in certain areas of Ethiopia recently, and over the
same time period, malaria has entered in to new areas where it was previously rare. This
raises the question, is the increase of maize cultivation partly responsible for the
increase in malaria?
One line of evidence is to look for an association between maize production and
malaria incidence at different geographically dispersed sites (Kebede et al. 2005). The
data set "malaria vs maize.csv" contains information on several high-altitude sites in
Ethiopia, with information about the level of cultivation of maize (low, medium or high
in the variable maize_yield) and the rate of malaria per 10,000 people
(incidence_rate_per_ten_thousand).

a. Plot a multiple histogram to show the relationship between level of maize

production and the incidence of malaria.

8
 b. ANOVA is a logical choice of method to test differences in the mean rate of
malaria between sites differing in level of maize production. Calculate the
standard deviation of the incidence rate for each level of maize yield. Do
these data seem to conform to the assumptions of ANOVA? Describe any
violations of assumptions you identify.

c. Compute the log of the incidence rate and redraw the multiple histograms
for different levels of maize yield. Calculate the standard deviation of the log
incidence rate for each level of maize yield. Does the log-transformed data
better meet the assumptions of ANOVA than did the untransformed data?

d. Test for an association between maize yield and malaria incidence.

3. Animals that are infected with a pathogen often have disturbed circadian rhythms. (A
circadian rhythm is an endogenous daily cycle in a behavior or physiological trait that
persists in the absence of time cues.) Shirasu-Hiza et al. (2007) wanted to know whether
it was possible that the circadian timing mechanism itself could have an effect on
disease. To test this idea they sampled from three groups of fruit flies: one "normal",
one with a mutation in the timing gene tim01, and one group that had the tim01
mutant in a heterozygous state. They exposed these flies to a dangerous bacteria,
Streptococcus pneumoniae, and measured how long the flies lived afterwards, in days.
The date file "circadian mutant health.csv" shows some of their data.

a. Plot a histogram of each of the three groups. Do these data match the
assumptions of an ANOVA?

b. Use a Kruskal-Wallis test to ask whether lifespan differs between the three
groups of flies.